Project Summary Bacterial resistance to l3-lactam antibiotics continues to become more prevalent and more clinically important. A large part of the resistance can be understood and investigated experimentally in terms of the chemistry of the interactions of R-lactam antibiotics with the active sites of two groups of bacterial enzymes, the Illactamases on one hand, which catalyze the hydrolysis of the antibiotics, and the D-alanyl-D-alanine transpeptidase/carboxypeptidases on the other, which catalyze the synthesis and maintenance of the peptide cross-links of bacterial cell walls, and which are inhibited by 13-lactam antibiotics. There is now good reason to believe that all of these l-lactam binding sites have much in common. An understanding of the structure and function of these sites and of the relationship between them is fundamental to future antibiotics design - both 1-lactam and otherwise. The object of the proposed research is to explore further the chemical functionality and the substrate binding properties of a series of these active sites, using a number of modified substrates, novel inhibitors and potential effedtors. Particular focus will be on the development of novel inhibitors of the (1lactamases and the extension of these inhibitors to DD-peptidases. The specificity of the DD-peptidases for peptidoglycan structural motifs will be examined closely. Crystal structures will be used in conjunction with molecular modeling to interpret the results obtained and apply them to further inhibitor design. In silico and in vitro screening methods will also be used with the OD-peptidases to obtain new lead compounds. These studies will lead to new insight into the chemistry of R-lactamase and transpeptidase active sites, and thus to new directions in antibiotic design.